20-substituted steroids

ABSTRACT

A compound selected from the group consisting of a compound of the formula                    
     wherein R 1  and R 2  are individually selected from the group consisting of alkyl of 1 to 12 carbon atoms and aralkyl of 7 to 15 carbon atoms or taken together form a saturated heterocycle of 5 to 6 ring members optionally having a second ring heteroatom selected from the group consisting of sulfur, oxygen and nitrogen, R 3  is an α-alkyl of 1 to 8 carbon atoms, n is an integer from 2 to 15, R 4  is alkyl of 1 to 12 carbon atoms, R 5  is selected from the group consisting of hydrogen, alkyl of 1 to 12 carbon atoms and acyl of an organic carboxylic acid of up to 12 carbon atoms and the wavy lines indicate that the 17- and 20-asymmetrical centers are independent of the absolute R and S configurations and their non-toxic, pharmaceutically acceptable acid addition salts useful for controlling fertility in male warm-blooded animals.

OBJECTS OF THE INVENTION

It is an object of the invention to provide the novel steroids offormula I and their non-toxic, pharmaceutically acceptable acid additionsalts and a novel process and novel intermediates for their preparation.

It is another object of the invention to provide novel compositions anda method for controlling fertility in male warm-blooded animals.

These and other objects and advantages of the invention will becomeobvious from the following detailed description.

THE INVENTION

The novel 20-substituted steroids of the invention are selected from thegroup consisting of a compound of the formula

wherein R₁ and R₂ are individually selected from the group consisting ofalkyl of 1 to 12 carbon atoms and aralkyl of 7 to 15 carbon atoms ortaken together form a saturated heterocycle of 5 to 6 ring membersoptionally having a second ring heteroatom selected from the groupconsisting of sulfur, oxygen and nitrogen. R₃ is an α-alkyl of 1 to 8carbon atoms, n is an integer from 2 to 15, R₄ is alkyl of 1 to 12carbon atoms, R₅ is selected from the group consisting of hydrogen,alkyl of 1 to 12 carbon atoms and acyl of an organic carboxylic acid ofup to 12 carbon atoms and the wavy lines indicate that the 17- and20-asymmetrical centers are independent of the absolute R and Sconfigurations and their non-toxic, pharmaceutically acceptable acidaddition salts.

Examples of R₁, R₂, R₄ and R₅ as alkyl of 1 to 12 carbon atoms aremethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl,n-hexyl, 2-methyl pentyl, 2,3-dimethyl-butyl, n-heptyl, 2-methylhexyl,2,2-dimethylpentyl, 3,3-dimethyl-pentyl, 3-ethyl-pentyl, n-octyl,2,2-dimethylhexyl, 3,3-dimethylhexyl, 3-methyl-3-ethylpentyl, nonyl,2,4-dimethylheptyl and n-decyl, preferably methyl, ethyl or isopropyl.When R₁ and R₂ is aralkyl of 7 to 15 carbon atoms, it is preferablybenzyl or phenethyl.

Examples of R₁ and R₂ forming with the nitrogen to which they are linkeda saturated heterocycle with 5 or 6 ring members optionally containinganother hetero ring atom chosen from oxygen, nitrogen and sulfur arepiperidino, morpholino, thiomorpholino, piperazino and pyrrolidino.

Examples of R₃ as alkyl of 1 to 8 carbon atoms are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl,2-methyl-phenyl, 2,3-dimethyl-butyl, n-heptyl, 2-methylhexyl,2,2-dimethylpentyl, 3,3-dimethyl-pentyl and 3-ethylpentyl, preferablymethyl.

Examples of acyl of an organic carboxylic acid of up to 12 carbon atomsare acetyl, propionyl, butyryl, benzoyl, valeryl, hexanoyl, acryloyl andcrotonoyl as well as formyl.

Examples of acids for the formation of non-toxic, pharmaceutically acidaddition salts are hydrochloric acid, hydrobromic acid, nitric acid,sulfuric acid, phosphoric acid, acetic acid, formic acid, propionicacid, benzoic acid, maleic acid, fumaric acid, succinic acid, tartaricacid, citric acid, oxalic acid, glyoxylic acid, aspartic acid, alkanesulfonic acids such as methane or ethane sulfonic acid, arylsulfonicacids such as benzene or p-toluene sulfonic acid and arylcarboxylicacids. The addition salts of hydrochloric acid are preferred.

Among the preferred compounds are those of formula I wherein n is 2 andthose of the formula

wherein R₁ and R₂ are defined above and their non-toxic,pharmaceutically acceptable acid addition salts.

Specific preferred compounds of formula I are (20R) (8α,9β,13α,14β,17α)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol,(20S)(8α,9β,13α,14β,17α)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol,(20R)(8α,9β,13α,14β,17β)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol,(20S) (8α,9β,13α,14β,17β)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol,as well as their acid addition salts. Most preferred is (20S)(8α,9β,13α,14β,17α)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-oland its acid addition salts.

The novel process of the invention for the preparation of the compoundsof formula I comprises subjecting a compound of the formula

wherein R₃ is defined as above to an acylation agent or alkylation agentto obtain a compound of the formula

wherein R₃ is defined as above and R′₅ is alkyl of 1 to 12 carbon atomsor acyl of an organic carboxylic acid of up to 12 carbon atoms, reactinga compound of formula II or IIA with a cyanidation agent to form acompound of the formula

in which R₃ and R₅ have the same meanings as above and in which the wavyline indicates that the product is presented in the form of purestereoisomers (17α-OH, 17β-CN) or (17α-CN, 17β-OH) or in the form of amixture thereof, subjecting the latter to a dehydration reaction toobtain a compound of the formula

in which R₃ and R₅ have the above meanings, reducing the 16-17 doublebond to obtain a compound of the formula

wherein the wavy line indicates that the —CN is in position 17α or 17β,or in the form of a 17α and 17β mixture, and R₃ and R₅ have the abovemeanings, reacting the latter with an organometal reagent of R₄ asdefined above, then to the action of an acid hydrolysis agent to obtaina compound of the formula

wherein R₃, R₄ and R₅ have the above meanings and in which the wavy lineindicates that the —COR₄ is in position 17α or 17β, or in the form of a17α and 17β mixture, reacting the latter with a hydroxyl-amine salt toobtain a compound of the formula

wherein R₃, R₄ and R₅ have the above meanings and in which the wavy lineindicates that —C(R₄)═N—OH is in 17α or 17β position or in the form of a17α and 17β mixture, and the oxime is in the syn or anti position, or inthe form of a syn and anti mixture, reducing the oxime to obtain acompound of the formula

wherein the wavy line indicates that —NH₂ is in 20R or 20S position orin the form of a 20R and 20S mixture, and R₃, R₄ and R₅ have the abovemeanings, reacting the latter with an acyl halide of the formula

X—CO—(CH₂)_(n)′—NR₁R₂

wherein X is halogen, R₁ and R₂ are as defined above, n′ is equal ton−1, n being defined as above, then, optionally to a selectivehydrolysis in the 3-position of the diacylated compound formed to obtaina compound of the formula

wherein the wavy lines, R₁, R₂, R₃, R₄, R₅ and n′ have the abovemeanings, reducing the keto group of the said amide, and optionally toone or more of the following reactions in any order:

acylation in position 3,

alkylation in position 3,

saponification of acyloxy in position 3,

separation of the different stereoisomers,

salification with an organic or mineral acid to obtain the compound offormula I.

The acylation agent is preferably a carboxylic acid derivative, forexample acid chloride or an anhydride in the presence of a base such aspyridine. The optional alkylation is carried out by the usual methodswith an alkylation agent such as preferably an alkyl halide like alkyliodide or alkyl sulfate.

The cyanidation agent is preferably sodium or potassium cyanide and thecyanidation reaction is preferably carried out in a lower alcohol suchas methanol in the presence of acetic acid. The dehydration reaction canbe carried out using a dehydration agent such as phosphorus oxychloridein pyridine.

The reduction of the 16-17 double bond can be carried out either bycatalytic hydrogenation with the hydrogenation agent being hydrogen inthe presence of catalysts such as palladium on charcoal, or a rhodiumreagent such as Wilkinson reagent, or by the action of sodiumborohydride in ethanol, or by the action of magnesium in methanol. Thisreduction is either stereospecific and allows the CH-substituent to beobtained in position 17α or in position 17β, or it is non-stereospecificin which a mixture of stereoisomers (17α+17β) is obtained which isoptionally separated by standard methods such as crystallization orchromatography.

The organometal reagents which are derivatives of R₄ are standardreagents such as an organolithium compound (R₄—Li), an organomagnesiumcompound (R₄—Mg—X) with X being halogen chosen from Cl, Br and I,preferably Br.

The acid hydrolysis reaction which follows the reaction with theorganometal allows the intermediate imine formed to be hydrolyzed whichhydrolysis is carried out under standard conditions for imine hydrolysisin an acid medium such as hydrochloric acid, oxalic acid or acetic acid.

Formation of the oxime of formula VII is preferably carried out by theaction of hydroxylamine hydrochloride in the presence of a base such aspyridine, sodium hydroxide or sodium carbonate.

The reduction of the product of formula VII can be carried out bydifferent methods such as catalytic hydrogenation with, as hydrogenationreagent, hydrogen in the presence of catalysts such as palladium oncharcoal or platinum dioxide, by the action of zinc in an acetic medium,by sodium in an alcohol such as ethanol or n-propanol, or also by theaddition of diborane in diglyme. This reduction is either stereospecificand allows the product in position 20R or in position 20S to beobtained, or it is non-stereospecific wherein a mixture of 20R+20Sstereoisomers is obtained which is optionally separated by standardmethods such as crystallization or chromatography.

The condensation of the compound of formula X—CO—(CH₂)_(n)′—NR₁R₂ inwhich X is halogen chosen from Cl, Br and I and n′, R₁ and R₂ are asdescribed previously with the compound of formula VIII is carried out ina basic medium, preferably in an aprotic dipolar solvent such asdimethylformamide (DMF). The reaction is preferably carried out in atriethylamine/dimethylformamide (TEA/DMF) medium.

The selective hydrolysis of the O-acyl compound which is optionallyformed intermediately is carried out under the usual conditions using anagent which can be an alkaline base such as sodium hydroxide orpotassium hydroxide in a lower alcohol such as methanol or ethanol.

The reduction of the keto group of the amide of formula IX is carriedout for example by means of a metal hydride such as lithium aluminumhydride. (AlLiH₄) in an aprotic polar solvent such as tetrahydrofuran(THF) or ether or by means of alkali metal borohydrides such as sodiumborohydride (NaBH₄) in the presence of acids such as acetic acid.

If desired and if necessary, the acylation or alkylation reactions ofthe 3 —OH group are carried out by the methods as described previously.The optional saponification reaction is preferably carried out in thepresence of an alkali metal base such as sodium hydroxide or potassiumhydroxide, potassium terbutylate or lithium acetylide in ethylene amine.The saponification reaction takes place preferably in a lower alcoholsuch as methanol or ethanol.

The optional separation of the different stereoisomers is carried out bystandard methods of crystallization or chromatography. The salificationwith an acid is carried out under the usual conditions preferably withhydrochloric acid for example in an ethereal solution.

During the action of a cyanidation agent leading to the product offormula III, with an organometal reagent leading to the product offormula VI or of the hydroxylamine salt leading to the product offormula VII, a product of formulae III, VI or VII can be obtained inwhich the acyloxy group is hydrolyzed.

The invention extends to a process as defined previously in which theproduct of formulae III, VI or VII in which the acyloxy group has beenhydrolyzed, is optionally reacylated.

The products of formulae V, VI, VII, VIII and IX are optionally obtainedin the form of a mixture of stereoisomers which can optionally besubjected to operations which separate these stereoisomers. Theinvention extends to a process as defined previously in which thedifferent stereosisomers obtained during preparation processes of theproducts of formulae V, VI, VII, VIII and IX are optionally separated.

The compositions of the invention for controlling fertility in malewarm-blooded animals are comprised of an amount of a compound of formulaand its non-toxic, pharmaceutically acceptable acid addition saltssufficient to control fertility and an inert pharmaceutical carrier. Thecompositions may be in the form of tablets, dragees, capsules, granules,suppositories, gels, creams, implants, microspheres, patches andinjectable solutions or suspensions or pessaries and particularlyvaginal pessaries.

Examples of suitable inert pharmaceutical carriers are talc, gum arabic,lactose, starch, magnesium stearate, cocoa butter, aqueous ornon-aqueous vehicles, fatty substances of animal or vegetable origin,paraffin derivatives, glycols, various wetting, dispersing oremulsifying agents and preservatives.

The compositions of the invention have 1) a strong affinity for theSigma receptors (see the pharmacological tests) or 2) an activityvis-à-vis the influx of the calcium into the spermatozoid. The resultsof tests show that among the products which fix to the Sigma receptors,certain act by stimulating the influx of calcium into the spermatozoidand others by inhibiting the influx of calcium stimulated or not byprogesterone, a molecule described as binding to the Sigma receptor.

The products of formula I having an agonist activity stimulate theinflux of calcium into the spermatozoid. The corresponding compositionsof the invention can be used in the treatment of certain forms ofsterility characterized by an insufficient fertilizing power of thespermatozoids.

The products of formula I having an antagonist activity inhibit theinflux of calcium into the spermatozoid. The corresponding compositionsof the invention are therefore of use in controlling the acrosomialreaction and consequently affect the fertilizing power of thespermatozoid. They can therefore be used, as a contraceptive and inparticular as a male contraceptive.

They can also be used in the veterinary domain as a male contraceptivein domestic animals (dogs, cats . . . ) or to limit the proliferation ofany pests, in particular rodents or pigeons.

The method of the invention for controlling fertility in malewarm-blooded animals comprises administering to male warm-bloodedanimals an amount of a compound of formula I or its acid addition saltsin an amount sufficient to control fertility. The compounds may beadministered orally, rectally or parenterally or locally, particularlyfor a woman, for example by percutaneous route or by injectionparticularly in the veterinary field and the usual daily dose is 0.10 to13.5 mg/kg depending on the specific compound, method of administrationand the condition treated.

The compound of formula II is available by the processes described byHutchinson et al., Tetrahedron Letters, 1985, Vol. 26(15), pp. 1819-1822and Smith et al, J. Am. Chem. Soc., 1966, pp. 3120-3128.

The intermediates of formulae II_(A), III, IV, V, VI, VII, VIII and IXare novel and a part of the invention with the exception of compounds offormula II_(A) in which R′₅ is an alkyl group containing at most 12carbon atoms.

In the following examples, there are described several preferredembodiments to illustrate the invention. However, it should beunderstood that the invention is not intended to be limited to thespecific embodiments.

EXAMPLE 1 (20S)(8α,9β,13α,14β,17α)20-((dimethylamino)-ethyl)-amino-19-nor-Δ^(1,3,5(10))-pregnatrien-3-olStage A 8α,9β,13α,14β 3-acetyloxy-Δ^(1,3,5(10))-estratrien 17-one

33.5 ml of acetic anhydride were added to a suspension of 33.3 g ofantipodal estrone (preparation as described in Hutchinson et al.,Tetrahedron Letters, 1985, Vol. 26(15), pp. 1819-1822) in 67 ml ofpyridine. A slightly exothermic dissolution was observed with thetemperature rising from 18° C. to 32° C. The mixture was stirred for 18hours at 18±2° C. and then was poured into a mixture of ice-cooled water(660 ml)/22° Bé hydrochloric acid (76 ml). After crystallization, thesuspension stood for 1 hour, was filtered, washed with water and driedto obtain 38.7 g of the expected crude product which was purified by hotand cold crystallization from 83 ml of absolute alcohol, followed bytreatment with L2S activated charcoal. After filtration and drying, 32.7g of the desired product were obtained with a melting point of 128° C.

Stage B (8α,9β,13α,14β,17β)3-(acetyloxy)-17-hydroxy-Δ^(1,3,5(10))-estratrien-17-carbonitrile

91.6 g of potassium cyanide were introduced under an inert gas into asolution of 32.7 g of estrone acetate of Stage A in 654 ml of methanoland 167 ml of acetic acid, and the mixture was stirred for 16 hours atambient temperature. Then, 330 ml of an ice-water mixture were added tothe suspension and after significant crystallization had been observed,the reaction medium was poured into 3 liters of ice-cooled water,followed by filtering and washing with water. The undried crude productwas dissolved in 1.2 liters of ethyl acetate and the organic phase waswashed, dried, filtered and concentrated until crystallization. Aftercooling down to −10° C. for 1 hour, filtering was carried out followedby washing and drying to obtain 27.2 g of the expected product meltingat 198-200° C.

Stage C(8α,9β,13α,14β)-Δ^(1,3,5(10))16-estra-tetraene-3-ol-17-carbonitrile

27.2 g of the product of Stage B in 82 ml of pyridine and 25 ml ofphosphorus oxychloride were refluxed for 4 hours and then, the reactionmedium was cooled to 20° C. and poured over 450 ml of crushed ice. Afteran exothermic precipitation had been observed, sulfuric acid diluted to⅕ was added to obtain a pH close to 1. Extraction was carried out withethyl acetate, followed by washing with water, then with a solution ofsodium bicarbonate, drying, filtering and evaporating to dryness underreduced pressure. The residue was dissolved in 60 ml of ethanol and thenthe solution was stirred for 1 hour at 0° C., followed by filtering anddrying to obtain 17.7 g of the expected product melting at 120° C.

Stage D(8α,9β,13α,14β,17α)-Δ^(1,3,5(10))-estratriene-3-ol-17-carbonitrile

1.425 liters of hydrogen were introduced over 14 minutes under nitrogeninto a suspension of 17.7 g of the product of Stage C in 354 ml of ethylacetate and 8.85 g of 10% palladium hydroxide on charcoal and themixture was stirred for 30 minutes. After filtering and evaporating todryness under reduced pressure, the dry extract was dissolved in 90 mlof ethanol, stirred for one hour at a temperature of −10° C., followedby separation and drying to obtain 15.35 g of the expected productmelting at 144.5° C. and having a specific rotation of [α]_(D)=−100°(c=1% in CHCl₃).

Stage E (8α,9β,13α,14β,17α)-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol-20-one

121 ml of methyl iodide were added at reflux over one hour to a mixtureof 46 g of magnesium turnings in 307 ml of benzene and 307 ml of etherunder inert gas. The reaction medium was refluxed for 30 minutes and asolution, prepared extemporaneously, of 15.35 g of the product of StageD in 154 ml of benzene and 154 ml of ether, was introduced. The mixturewas stirred at reflux for 93 hours and after reflux was stopped, thesuspension was slowly poured into a water/ice mixture, and 340 ml ofacetic acid (pH=4) were added. After concentration, separation wascarried out followed by washing and drying to obtain 13.7 g of crudeproduct which was purified in 840 ml of acetone and 0.6 g of 3SAactivated charcoal, filtered, concentrated to 5 vol., stirred for onehour at a temperature of −10° C., separated and dried to obtain 12.2 gof the expected product melting at 248° C. and having a specificrotation of [α]_(D)=−156.6° (c=0.5% in CHCl₃).

Stage F (8α,9β,13α,14β,17α)20-hydroxyimino-l9-nor-Δ^(1,3,5(10))-pregnatrien-3-ol

4.5 g of hydroxylamine hydrochloride were added to a solution, undernitrogen, of 10 g of the product of Stage E in 100 ml of pyridine andthe mixture was heated to 80-85° C. for 90 minutes. Then, 310 ml ofdemineralized water were added and crystallization of the product wasobserved. The crystals were separated and crystallized from 120 ml ofethanol at reflux. Another 75 ml of demineralized water were added andsignificant crystallization of the product was observed. The mixture wasstirred for 30 minutes at 0C, followed by separation and drying toobtain 9.45 g of the expected product melting at 234° C.

Stage G (20S)(8α,9β,13α,14β,17α)20-amino-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol

7.35 g of the product of Stage F in 550 ml (+368 ml for rinsing) ofacetic acid were added to a suspension of 2.94 g of platinum dioxide in304 ml of acetic acid and hydrogenation was carried out with a totalabsorbed volume of 1075 ml of hydrogen over 6 hours and 30 minutes.

Obtaining the Hydrochloride

After filtration, concentration under reduced pressure was carried outuntil a dry extract was obtained which was taken up in an acid medium ofa mixture of 4.15. ml of hydrochloric acid in 53.5 ml of ethanol and 1.2ml of water. 92 ml of ether were added to the solution which was stirredat 0° C. for 1 hour, followed by separation and drying. The crudehydrochloride was crystallized by dissolution at reflux in 50 ml ofethanol with 0.5% hydrochloric acid and stirring for 1 hour at 0+5° C.followed by separation.

Obtaining the Base

140 ml of demineralized water were slowly added hot to a solution of thesaid hydrochloride purified in a basic mixture of 11 ml oftriethylamine, 64 ml of ethanol and 27 ml of water at reflux.Crystallization was observed and the mixture was stirred for 1 hour at0+5° C., followed by separation and drying to obtain 3.77 g of the crudebase which was purified by dissolution at reflux in 120 ml of ethanol,concentration at normal pressure and under nitrogen to a volume of 40ml, stirring for 1 hour at 0+5° C., separation and drying to obtain3.285 g of the expected product melting at 235° C.

Stage H (20S)(8α,9β,13α,14β,17α)2-dimethylamino-N-(19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol-20-yl)-acetamide

12 g of the hydrochloride of N,N dimethyl-glycine chloride were addedrapidly under inert gas to a solution of the product of Stage G in 91.5ml of dimethylformamide and 28.4 ml of triethyl-amine, obtained at 80°C., then cooled to +5° C., followed by stirring for 3 hours. The mixturewas poured into a saturated solution of 370 ml of sodium bicarbonate in550 ml of ice+water. The mixture was stirred for 1 hour followed byextraction 3 times with 100 ml of dichloromethane and washing withwater, then with a solution of sodium bicarbonate, then with a solutionof salt water. The combined organic solutions were concentrated underreduced pressure until a dry extract of 6 g was obtained.

The residue was taken up under inert gas in 37 ml of methanol and 11 mlof 5N sodium hydroxide, followed by stirring for 1 hour until totaldissolution. 220 ml of demineralized water were added slowly and carbondioxide (pH 8) was bubbled through. 14.7 ml of triethylamine were addedand the mixture was stirred for 15 minutes, followed by extraction with250 ml, then 100 ml of dichloromethane, washing 5 times with 100 ml ofwater. The organic solution was dried, treated on 3SA activatedcharcoal, filtered, concentrated under reduced pressure until a dryextract (oil) was obtained which was crystallized by two entrainmentswith ethanol to obtain 6.15 g of the expected crude product which wasdissolved at reflux in 80 ml of ethanol, treated with L2S activatedcharcoal, filtered and concentrated at normal pressure to a volume of 40ml. Crystallization was observed and 10 ml of water were added. Themixture was stirred for 1 hour at 0°+5° C., followed by separation anddrying to obtain 3.03 g of the expected product melting at 226° C.

Stage I (20S)(8α,9β,13α,14β,17α)20-(((dimethylamino)-ethyl)-amino-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol

3.03 g of the product of Stage H were added at a temperature of 20° C.to a suspension under inert gas of 1.845 g of lithium aluminum hydrideand 5.25 g of aluminum chloride in 131 ml of tetrahydrofuran and themixture was refluxed for 24 hours. After cooling to 0+5° C., 20 ml ofethyl acetate were added and then 100 ml of a saturated solution ofsodium chloride were added. The suspension was filtered, thensuccessively taken up in a water/6N HCl mixture (80 ml/50 ml), filtered,taken up in 60 ml of 60% ethanol and 8 ml of triethylamine and filtered.Water was added to the solution and precipitation was observed.Extraction was carried out with dichloromethane, followed by washing,drying, treating on L2S activated charcoal and concentration underreduced pressure until a dry extract of 1.9 g was obtained. This dryextract was dissolved at reflux in 60 ml of ethyl acetate and 4 drops oftriethylamine for 15 minutes and then the solution was concentratedunder reduced pressure to a volume of 30 ml, cooled over 1 hour to 0+5°C., separated and dried to obtain 1.03 g of the expected product meltingat 177° C. and having a specific rotation of [α]_(D)=−80.6° (c=0.5% inEtOH). Analysis: C₂₄H₃₈ON₂; molecular weight=370.56

C H N % calculated: 77.78 10.34 7.56 % found 77.9 10.3 7.4

Pharmacological Tests

Method

Preparation of the Human Spermatozoids

The human sperm originated from healthy donors. The mobile spermatozoidswere separated by centrifugation on a Percoll gradient (47.5-95%) andthen resuspended in a hypertonic BWW medium containing: 166 mM of NaCl,5 mM of KCl, 1.3 mM of CaCl₂, 1.2 mM of KH₂PO₄, 1.2 mM of MgSO₄, 5.5 mMof glucose, 21 mM of sodium lactate, 0.25 mM of sodium pyruvate, 25 mMof NaHCO₃, 20 mM of Hepes and 0.8% of HSA (410 mosm/liter), pH 7.4 atambient temperature.

Measurement of the Intracellular Calcium

The mobile spermatozoids were incubated for a minimum of 2 hours in theBWW/HSA capacitating medium and were then incubated at a concentrationof 5-10×10⁶/ml with Fura2-AM to a final concentration 2 μM at 37° C. for45 minutes. After washing by centrifugation at 600 g for 10 minutes in.BWW without HSA, the spermatozoids were suspended at a concentration of4×10⁶/ml. The florescence signal was measured at 37° C. using aspectrofluorimeter at excitation wave lengths of 340 and 380 nm (PTIM2001-Kontron) or at 340, 360 and 380 nm (Hitachi F 2000—B. Braun ScienceTec.). The fluorescence emission was recorded at 505 nm. Theprogesterone or the products to be tested, dissolved in absoluteethanol, were added to the incubation medium at a final concentration of0.1% of ethanol.

When an antagonistic effect of the progesterone was sought, the productwas added to the medium 2 minutes before the progesterone. At the end ofthe each dosage, 5 μM of ionomycin were added to the sample to measurethe maximum fluorescence signal. Then, the spermatozoids werepermeabilized with 0.05% of Triton X-100 and 10 mM of EGTA were added(pH 9.5) to measure the minimum fluorescence signal. These valuesallowed the intracellular concentration of calcium ([Ca²⁺]i) to becalculated by the method described by Grunkiewicz et al. 1985, J. Biol.Chem., Vol. 260, pp. 3440-3450). The results of the intracellularcalcium concentra-tions were expressed relative to the basal level whichwas arbitrarily set equal to 1.

Sigma Receptor: Measurement of the Relative Bond Affinity

The relative bond affinity was evaluated for preparations of rat brainand testicle membranes.

Preparation of the Membranes

Male Sprague-Dawley rats originating from Iffa Credo and weighingapproximately 200 g were used. The animals were scarified bydecapitation and the brain and testicles were removed and homogenized in10 to 25 volumes of 50 mM Tris-HCl buffer (pH 7.7) at 4° C. using anUltrathurax. The homogenates were centrigued at 30,000 g for 15 minutesat 4° C. and the pellets were washed 3 times by resuspension in the samebuffer and centrifugation under the same conditions. The membranesobtained in this way were stored at −80° C.

Incubation

The marker of the sigma receptors used was ³H PPP(propyl-3-(propyl-3-(3-hydroxyphenyl)-piperidine) of NEN having aspecific activity of 3404 GBq/mmol. The membranes were suspended in the50 mM Tris-HCl buffer, pH 8.0 to obtain a concentration of proteins ofapproximately 0.6 mg/ml for the testicles and 1 mg/ml for the brain.Aliquots of the homogenate were incubated for 90 minutes at 25° C. in atotal volume of 0.5 ml with 3 nM of ³H PPP in the presence of increasingconcentrations of reference product (haloperidol) or of the products tobe tested. At the end of the incubation, the 3H PPP bound to themembranes was separated from the free ³H PPP by rapid filtration onWhatman GF/C filters pre-treated beforehand with 0.05% ofpolyethyleneimine. The precipitate was washed twice with 5 ml ofTris-HCl buffer and the radioactivity was counted after the addition of20 ml of Aqualyte scintillating liquid (Baker).

Calculation of the Relative Bond Affinity (RBA)

The following two curves were drawn: percentage of bound tritiatedmarker 100×B/BO as a function of the logarithm of the concentration ofunlabelled reference product or as a function of the logarithm of theconcentration of unlabelled test product. The straight line of thefollowing equation was determined:

I ₅₀=100(BO/BO+Bmin/BO)/2

i.e.

I ₅₀=100(1+Bmin/BO)/2=50(1+Bmin/BO)

BO=concentration of the bound tritiated marker in the absence of anyunlabelled product.

B=concentration of the bound tritiated marker in the presence of aconcentration X of unlabelled product.

Bmin=concentration of the bound tritiated marker in the presence of alarge excess of the unlabelled reference product (5,000 nM).

The intersections of the straight line I₅₀ and the curves allowed theevaluation of the concentrations of the unlabelled reference product(CH) and of the unlabelled test product (CX) which inhibited by 50% thespecific binding of the tritiated marker on the receptor. The relativebond affinity (RBA) of the test product was determined by the equation:

RBA=100(CH)/(CX).

The RBA of the haloperidol was arbitrarily set equal to 100.

PHARMACOLOGICAL TESTS 1- Relative bond affinity (RBA) for the Sigmareceptor Brain Testicle (rat) (rat) Ref: Haloperidol 100.0 100.0Progesterone 0.7 0.3 Estrone <0.06 <0.1 Product of Example 1 (product U)4.3 58.0

2—Measurement of Intracellular Calcium

Effect of progesterone at 10⁻⁵M after 2 minutes of pre-treatment withproduct U, at different doses 10⁻⁸M to 10⁻⁵M on [Ca²⁺]i Mean±SEM n=3

PROG. U10⁻⁸M + U10⁻⁷M + U10⁻⁶M + U10⁻⁵M + alone PROG. PROG. PROG. PROG.3.67 ± 0.97 3.33 ± 0.87 4.26 ± 1.93 2.63 ± 0.57 1.0 ± 0.0

The results were expressed relative to the basal level set equal to 1.Value of the basal level in the three experiments was 176.70±22.90 nM.

Antagonistic effect of the Product U at 10⁻⁵ M on the effect of PROG at10⁻⁵ M. Mean±SEM n=8

Pre-treatment 2 mn with PROG. 10⁻⁵M alone U10⁻⁵M alone U10⁻⁵M + PROG.10⁻⁵M 5.76 ± 0.84 0.95 ± 0.05 0.95 ± 0.05

The results were expressed relative to the basal level set equal to 1.

CONCLUSION

Effect on the Intracellular Calcium of Human Spermatozoids

Progesterone at the concentration of 10⁻⁵ M induced a transitoryincrease of the [Ca²⁺]i followed by a second phase where the [Ca²⁺]i wasslightly greater than the basal level. As for Product U at 10⁻⁵M, itcompletely antagonized the effect of progesterone when it was added tothe medium 2 minutes before the progesterone.

Relative Bond Affinity (RBA) for the Sigma Receptor

Product U and progesterone were capable of displacing ³H PPP. The RBA'scalculated using rat brain membranes had also been evaluated ontesticles and are given in the table. The differences observed betweenthe RBA's at the level of the brain and testicles could be explained bya different distribution of the various types of sigma receptor sitesbetween these two organs. Such products could therefore inhibit theacrosomial reaction (essential stage of fertilization) in the case ofantagonists such as Product U and could therefore be used as a malecontraceptive.

Various modifications of the compounds and method of the invention maybe made without departing from the spirit or scope thereof and it is tobe understood that the invention is intended to be limited only asdefined in the appended claims.

What is claimed is:
 1. A compound selected from the group consisting ofa compound of the formula

wherein R₁ and R₂ are individually selected from the group consisting ofalkyl of 1 to 12 carbon atoms and aralkyl of 7 to 15 carbon atoms ortaken together form a saturated heterocycle of 5 to 6 ring membersoptionally having a second ring heteroatom selected from the groupconsisting of sulfur, oxygen and nitrogen, R₃ is an α-alkyl of 1 to 8carbon atoms, n is an integer from 2 to 15, R₄ is alkyl of 1 to 12carbon atoms, R₅ is selected from the group consisting of hydrogen,alkyl of 1 to 12 carbon atoms and acyl of an organic carboxylic acid ofup to 12 carbon atoms and the wavy lines indicate that the 17- and20-asymmetrical centers are independent of the absolute R and Sconfigurations and their non-toxic, pharmaceutically acceptable acidaddition salts.
 2. A compound of claim 1 wherein n is
 2. 3. A compoundof claim 1 having the formula

wherein R₁ and R₂ have the definitions of claim 1 and their non-toxic,pharmaceutically acceptable acid addition salts.
 4. A compound of claim1 selected from the group consisting of (20R)(8α,9β,13α,14β,17α)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol,(20S)(8α,9β,13α,14β,17α)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol,(20R)(8α,9β,13α,14β,17β)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-ol,(20S)(8α,9β,13α,14β,17β20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-oland their non-toxic, pharmaceutically acceptable acid addition salts. 5.A compound of claim 1 which is (20S)(8α,9β,13α,14β,17α)20-[((dimethylamino)-ethyl)-amino]-19-nor-Δ^(1,3,5(10))-pregnatrien-3-oland its non-toxic, pharmaceutically acceptable acid addition salts.
 6. Aprocess for the preparation of a compound of claim 1 comprisingsubjecting a compound of the formula

wherein R₃ is defined as in claim 1 to an acylation agent or alkylationagent to obtain a compound of the formula

wherein R′₅ is alkyl of 1 to 12 carbon atoms or acyl of an organiccarboxylic acid of up to 12 carbon atoms, reacting a compound of formulaII or IIA with a cyanidation agent to form a compound of the formula

in which R₃ and R₅ have the same meanings as in claim 1 and in which thewavy line indicates that the product is presented in the form of purestereoisomers (17α-OH, 17β-CN) or (17α-CN, 17β-OH) or in the form of amixture thereof, subjecting the latter to a dehydration reaction toobtain a compound of the formula

in which R₃ and R₅ have the above meanings, reducing the 16-17 doublebond to obtain a compound of the formula

wherein the wavy line indicates that the —CN is in position 17α or 17β,or in the form of a 17α and 17β mixture, and R₃ and R₅ have the abovemeanings, reacting the latter with an organometal reagent of R₄ asdefined in claim 1, then to the action of an acid hydrolysis agent toobtain a compound of the formula

wherein R₃, R₄ and R₅ have the above meanings and in which the wavy lineindicates that the —COR₄ is in position 17α or 17β, or in the form of a17α and 17β mixture, reacting the latter with a hydroxyl-amine salt toobtain a compound of the formula

wherein R₃, R₄ and R₅ have the above meanings and in which the wavy lineindicates that —C(R₄)═N—OH is in 17α or 17β position or in the form of a17α and 17β mixture, and the oxime is in the syn or anti position, or inthe form of a syn and anti mixture, reducing the oxime to obtain acompound of the formula

wherein the wavy line indicates that —NH₂ is in 20R or 20S position orin the form of a 20R and 20S mixture, and R₃, R₄ and R₅ have the abovemeanings reacting the latter with an acyl halide of the formulaX—CO—(CH₂)_(n)′—NR₁R₂ wherein X is halogen, R₁ and R₂ are as defined inclaim 1, n′ is equal to n−1, n being defined as in claim 1, then,optionally to a selective hydrolysis in the 3-position of the diacylatedcompound formed to obtain a compound of the formula

wherein the wavy lines, R₁, R₂, R₃, R₄, R₅ and n′ have the abovemeanings, reducing the keto group of the said amide, and optionally toone or more of the following reactions in any order: acylation inposition 3, alkylation in position 3, saponification of acyloxy inposition 3, separation of the different stereoisomers, salification withan organic or mineral acid to obtain the compound of formula I.